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. 2009 Oct;23(10):1646–1656. doi: 10.1210/me.2009-0147

Epidermal Growth Factor-Induced GnRH-II Synthesis Contributes to Ovarian Cancer Cell Invasion

Song Ling Poon 1, Gareth T Hammond 1, Peter C K Leung 1
PMCID: PMC5419146  PMID: 19608641

Abstract

GnRH-II modulates ovarian cancer cells invasion and is expressed in normal ovary and ovarian epithelial cancer cells; however, the upstream regulator(s) of GnRH-II expression in these cells remains unclear. We now demonstrate that epidermal growth factor (EGF) increases GnRH-II mRNA levels in several human ovarian carcinoma cell lines and up-regulates GnRH-II promoter activity in OVCAR-3 cells in a dose-dependent manner, whereas an EGF receptor inhibitor (AG148) abolishes EGF-induced increases in GnRH-II promoter activity and GnRH-II mRNA levels. EGF increases the phosphorylation of cAMP-responsive element-binding protein (p-CREB) and its association with the coregulator, CCAAT/enhancer binding protein β, whereas blocking the EGF-induced ERK1/2 phosphorylation with MAPK inhibitors (PD98059/U0126) markedly reduced these effects. Moreover, depletion of CREB using small interfering RNA attenuated EGF-induced GnRH-II promoter activity. Chromatin immunoprecipitation assays demonstrated that EGF induces p-CREB binding to a cAMP responsive-element within the GnRH-II promoter, likely in association with CCAAT/enhancer binding protein β, and mutagenesis of this cAMP responsive-element prevented EGF-induced GnRH-II promoter activity in OVCAR-3 cells. Importantly, GnRH-II acts additively with EGF to promote invasion of OVCAR-3 and CaOV-3 cells, but not SKOV-3 cells that express low levels of GnRH receptor (GnRHR). Treatment with GnRHR small interfering RNA also partially inhibited the EGF-induced invasion of OVCAR-3 and CaOV-3 cells. Furthermore, EGF treatment transiently increases GnRHR levels in OVCAR-3 and CaOV-3, which likely accentuates the effects of increase GnRH-II production on cell invasion. These results provide evidence that EGF is an upstream regulator of the autocrine actions of GnRH-II on the invasive properties of ovarian cancer cells.

GnRH-II induced by EGF via ERK1/2 and pCREB/C/EBPb signaling acts in an autocrine/paracrine manner to enhance ovarian cancer cell invasion.

The two GnRHs (GnRH-I and GnRH-II) and the GnRH receptor (GnRHR) have been detected in human ovarian surface epithelial cells and ovarian cancer cell lines (1), and these GnRH subtypes regulate the growth (2, 3, 4) and metastatic activity (5, 6) of ovarian cancer cells. Despite advances in our knowledge of the functional role of GnRH-II in ovarian cancer, the endocrine regulation of GnRH-II expression in ovarian cancer cells is poorly understood. In female reproductive tumor cell lines, studies of cell signaling have focused on the cAMP-mediated activation of protein kinase A and the subsequent phosphorylation-dependent activation of cAMP-responsive binding protein (CREB) (7). Elevated phosphorylated CREB (p-CREB) recruits the co-regulators, CREB binding protein (CBP) and CCAAT/enhancer binding protein β (C/EBPβ), and increases the cAMP responsive element (CRE)-dependent expression of the GnRH-II gene in a coordinated and temporally defined manner (7). Additional cis-regulatory regions, including a minimal promoter region that includes two enhancer elements (E-boxes); an E26 transformation specific (ETS)-like element in the untranslated exon 1 (8), and a nuclear factor-κB recognition site in the first intron (9) of the human GnRH-II gene, have also been identified.

In ovarian carcinoma cells, epidermal growth factor (EGF) and EGF-related peptides function as autocrine growth factors (10). The EGF receptor (EGFR) also plays a role in cancer cell biology and is a key therapeutic target in ovarian cancer (11). Classical EGFR signal transduction is initiated by ligand binding to its extracellular domain, which leads to a conformational change in the receptor and induces its homodimerization or heterodimerization with other EGFR family members (12, 13, 14). Ligand-induced EGFR dimerization allows trans-phosphorylation of specific tyrosine residues that serve as docking sites for intracellular signaling molecules (11), thereby stimulating the receptor’s intrinsic tyrosine kinase activity (15). Each EGFR is capable of recruiting a specific subset of adapter proteins and signaling molecules, such as Ras/Raf1/MAPK and phosphatidylinositol 3-kinase/Akt, which subsequently activate downstream mediators to stimulate cell proliferation, invasion, and angiogenesis (16, 17, 18). Gene amplification, genetic mutation, and altered transcription or translation result in aberrant EGFR expression that contributes to malignant transformation (19, 20) and poor prognosis and decreased therapeutic responsiveness in ovarian cancer patients (21, 22, 23). Thus, anticancer agents targeting the EGFR or its downstream signaling/target genes hold great promise.

In the present study, we sought to determine whether EGF regulates the expression of GnRH-II in ovarian cancer cells. Our results demonstrate that EGF treatment of OVCAR-3 cells increases GnRH-II promoter activity and GnRH-II mRNA levels through the autophosphorylation of EGFR and the activation of ERK1/2/p-CREB/C/EBPβ signaling. The stimulatory effect of EGF was observed in three human ovarian cancer cell lines (OVCAR-3, CaOV-3, and SKOV-3 cells), and this increase in GnRH-II by EGF promotes the EGF-induced invasiveness of ovarian cancer cells, suggesting that GnRH-II is a novel downstream target of EGF in ovarian cancer cell tumorigenicity.

Results

EGF stimulates GnRH-II expression in OVCAR-3, CaOV-3, and SKOV-3 cells

When treated with EGF, 2- to 3-fold increases in GnRH-II mRNA levels were observed in OVCAR-3 (Fig. 1A), CaOV-3 (Fig. 1B), and SKOV-3 (Fig. 1C) cells. Whereas EGF specifically induced the expression of GnRH-II in these cell lines, it had no effect on GnRH-I or GnRHR mRNA levels after 24 h treatment. However, by treating all three cell lines with EGF for 2, 4, 8, 16, and 24 h, we observed a modest increase in GnRHR protein levels after 8 h treatment in OVCAR-3 and CaOV-3 cells, but not in SKOV-3 cells (Fig. 1D). In addition, increased GnRHR levels returned to the pretreatment levels after 24 h in both OVCAR-3 and CaOV-3 cells (Fig. 1D). Using OVCAR-3 cells as our model, EGF treatment increased the activity of a GnRH-II promoter-driven luciferase reporter gene in a dose-dependent manner (Fig. 2A). Blocking downstream signaling by using the EGFR inhibitor, AG1478, abolished the EGF-induced GnRH-II promoter-driven luciferase reporter gene activity (Fig. 2B), as well as GnRH-II mRNA levels in OVCAR-3 cells (Fig. 2C), suggesting that the effect of EGF on GnRH-II regulation is EGFR dependent.

Fig. 1.

Fig. 1.

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EGF induces GnRH-II mRNA in ovarian cancer cell lines. OVCAR-3 cells (A), CaOV-3 cells (B), and SKOV-3 cells (C) were treated with 100 ng/ml of EGF for 24 h. Total RNA was isolated, and cDNA was used in real-time PCR to evaluate the effect of EGF on GnRH-II, GnRH-I, and GnRHR mRNA levels expressed as percentage over control (Ctrl) level. *, P < 0.05 compared with untreated control. D, OVCAR-3 cells, CaOV-3 cells, and SKOV-3 cells were treated with 100 ng/ml EGF for increasing times (2, 4, 8, 16, 24 h). Cells harvested were then subjected to Western blotting and probed for GnRHR. β-Actin was used as a normalization control.

Fig. 2.

Fig. 2.

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EGFR-dependent activation is required for the stimulation of GnRH-II expression in OVCAR-3 cells. A, OVCAR-3 cells were treated with increasing doses of EGF after transient transfection with a GnRH-II promoter-driven luciferase reporter gene construct together with a RSV-lacZ plasmid. B, A similar experiment was performed in which 10 μm EGFR inhibitor, AG1478, was pretreated for 30 min and then cotreated in the presence or absence of 100 ng/ml EGF in OVCAR-3 cells. Cell lysates were collected for luciferase assay and measurements of β-galactosidase activity as a control for transfection efficiency. Results are expressed as mean ± sem luciferase activity/β-galactosidase activity (i.e. GnRH-II promoter luciferase activity) of three independent experiments. C, In parallel experiments, total RNA was isolated after the administration of AG1478 in the presence or absence of 100 ng/ml EGF for 24 h and subjected for real-time RT-PCR to evaluate the effect of EGF on GnRH-II mRNA levels expressed as fold changes over control (Ctrl) levels in OVCAR-3 cells. *, P < 0.05 compared with untreated control.

EGF induces the MAPK pathway to enhance phosphorylation of CREB and its interaction with C/EBPβ upon EGFR activation

Treatment of OVCAR-3 cells with EGF induces phosphorylation of EGFR at tyrosine 992 and tyrosine 1045 (Fig. 3A) but not at tyrosine 1068 (data not shown). In addition, EGF very rapidly induces the phosphorylation of ERK1/2 with a maximum response at 15 min (Fig. 3B), suggesting that MAPK signaling may be involved in the regulation of GnRH-II expression in OVCAR-3 cells. The transcription factor CREB is a target of MAPK signaling (24) and is necessary for the regulation of GnRH-II gene expression (7). Western blot results indicate that CREB phosphorylation not only occurs very rapidly (within 10 min) but remains elevated for up to 8 h (Fig. 3C). Thus, we examined whether EGF-induced MAPK signaling results in the phosphorylation of CREB. We used a pharmacological inhibitor (PD98059) to block EGF-induced ERK1/2 signaling and examined the status of p-CREB (Fig. 3D). The results of these Western blotting experiments indicate that EGF rapidly induced CREB phosphorylation within 2 h, and that pretreatment with PD98059 attenuated EGF-induced phosphorylation of CREB in OVCAR-3 cells (Fig. 3D).

Fig. 3.

Fig. 3.

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EGF-activated ERK1/2 pathway is required for the phosphorylation of CREB and its interaction with C/EBPβ. A, OVCAR-3 cells were treated with 100 ng/ml of EGF for different time slots (5, 15, 30, 60 min) or (2, 4, 8, 16, 24 h). Cells harvested were then subjected to Western blotting and probed for phosphorylated EGFR (p-EGFR) at different tyrosines (992 and 1045). B and C, Nuclear lysates harvested from EGF-treated cells were subjected to Western blotting and probed for p-ERK1/2 and p-CREB, respectively. D, OVCAR-3 cells were pretreated with 20 μm PD98059 (selective MAPK inhibitor) for 30 min and then cotreated in the presence or absence of 100 ng/ml EGF for 2 h or 4 h. Nuclear cell lysates were collected, and the level of p-CREB was determined by Western blotting. Total EGFR (EGFR), total CREB (CREB), total ERK1/2 (ERK1/2), and β-actin were used as normalization control. E, OVCAR-3 cells were pretreated with 20 μm U0126 (selective MAPK inhibitor) for 30 min and then treated in the presence or absence of 100 ng/ml EGF for 2, 4, or 8 h. Nuclear cell lysates were collected and subjected to immunoprecipitation (IP) with p-CREB antibody, and the IPs were Western blotted with C/EBPβ antibody. The Western blot is representative of IPs from three independent experiments. Ctrl, Control.

Previously, we demonstrated that an increase of p-CREB recruits C/EBPβ and up-regulates GnRH-II transcription (7). Using immunoprecipitation, we have shown that the administration of 100 ng/ml EGF to OVCAR-3 cells enhances the association of p-CREB with C/EBPβ (Fig. 3E). More importantly, the EGF-induced interaction between p-CREB and C/EBPβ was markedly reduced upon pretreatment with U0126, a pharmacological agent that specifically inhibits phosphorylated ERK1/2 (p-ERK1/2) signal transduction pathways (Fig. 3E).

EGF-induced phosphorylation of CREB activates the cAMP responsive element (CRE) within the GnRH-II promoter

To determine whether EGF-induced p-CREB in OVCAR-3 cells targets the CRE region within the GnRH-II promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Cross-linked, sheared chromatin from EGF-treated OVCAR-3 cells was immunoprecipitated with p-CREB antibody, and the recovered DNA was subjected to PCR using primers specific to the CRE region (−860/−853) of the GnRH-II promoter. As shown in Fig. 4A, a 213-bp PCR product was amplified from p-CREB-immunoprecipitated DNA samples in OVCAR-3 cells treated with EGF, and this was already evident after 1 h, and is increased at 2–4 h of EGF stimulation (Fig. 4A). By contrast, little or no PCR product was observed with DNA recovered when control IgG was used for the immunoprecipitation or cells untreated with EGF. These data reveal a specific association between p-CREB at the CRE region of the GnRH-II promoter in OVCAR-3 cells after treatment with EGF and that this occurs in a temporally defined manner.

Fig. 4.

Fig. 4.

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EGF regulates the binding of p-CREB on CRE site in GnRH-II promoter. A, OVCAR-3 cells were treated with 100 ng/ml EGF for 1 h, 2 h, or 4 h, or were untreated [control (Ctrl)]. Cross-linked, sheared chromatin was immunoprecipitated (IP) with p-CREB, and recovered chromatin was subjected to PCR analysis using primers spanning the CRE region of the GnRH-II promoter. The IgG lanes are ChIPs performed using nonspecific IgG. An ethidium bromide-stained gel of PCR products shows a representative of ChIP analysis from three independent experiments. B, The efficiency of the siRNA was tested by Western blotting for CREB (67.5% knockdown) in OVCAR-3 cells. C, Cells were transfected with GnRH-II promoter-driven luciferase reporter gene construct together with 150 nm random siRNA controls (si-Ctrl) or siRNAs for CREB (si-CREB), respectively, and then treated with 100 ng/ml EGF for 24 h. D, OVCAR-3 cells were transfected with wild-type GnRH-II promoter-driven lucifease reporter gene construct or a 3-bp mutated CRE-GnRH-II promoter-driven luciferase reporter construct followed by 100 ng/ml EGF or 1 mm cAMP treatment. Cell lysates were assayed for luciferase activity and measurements of β-galactosidase activity as a control for transfection efficiency, the result of which are expressed as mean ± sem luciferase activity β-galactosidase activity (i.e. GnRH-II promoter luciferase activity) of three independent experiments.*, P < 0.05 compared with cells treated with a siRNA control. #, P < 0.05 compared with cells treated with specific CREB siRNA and followed by EGF treatment.

A specific small interfering RNA (siRNA) oligonucleotide was used to knock down endogenous CREB levels to verify its involvement in GnRH-II expression in OVCAR-3 cells (Fig. 4B). In this experiment, EGF induced GnRH-II promoter activity in cells cotransfected with control siRNA, whereas cotransfection with CREB-specific siRNA compromised this effect of EGF (Fig. 4C). To further verify that the CRE within the GnRH-II promoter is sufficient for EGF-regulated GnRH-II expression, we mutated 3 bp within the CRE (wild-type CRE: agacgtca; mutated CRE: agatacca) of the GnRH-II promoter-driven luciferase reporter gene. Transfection of the mutated reporter gene in OVCAR-3 cells resulted in a 35% decrease in basal GnRH-II promoter activity. Moreover, whereas treatment with 1 mm cAMP led to a significant increase in the wild-type GnRH-II promoter-driven luciferase reporter gene, it had no effect in cells transfected with the CRE-mutated version of the same reporter construct. In a parallel experiment, 100 ng/ml EGF induced the activation of the wild-type GnRH-II promoter, but this stimulation was reduced by 70% when we mutated the CRE within the GnRH-II promoter (Fig. 4D). These results indicate that the CRE we have examined is important for basal GnRH-II promoter activity, and that EGF exerts most of its effects through this particular cis-acting element to enhance the transcription of GnRH-II in OVCAR-3 cells.

EGF and GnRH-II act additively to enhance ovarian cancer cell invasion

As a strong mitogen, EGF enhances cell motility and induces secretion of proteolytic enzymes to increase the invasiveness in ovarian cancer cells (25, 26). To evaluate the effect of EGF-induced GnRH-II expression in ovarian cancer cells, OVCAR-3 cells, CaOV-3 cells, and SKOV-3 cells were treated with 100 ng/ml EGF or 10 nm GnRH-II for 24 h before an invasion assay. In these experiments, EGF-treated cells exhibit increased invasiveness as compared with their untreated controls. Interestingly, in the GnRH-II treated group, only OVCAR-3 cells (Fig. 5A) and CaOV-3 cells (Fig. 5B) showed 20-fold and 2-fold increases in their invasiveness, respectively, as compared with the controls, whereas SKOV-3 cells did not respond (Fig. 5C). When we used Western blotting to check the expression of the GnRHR in all three cell lines, we found that the expression of GnRHR in SKOV-3 cells is very much lower then in OVCAR-3 and CaOV-3 cells (Fig. 5D), and this suggests that ovarian cancer cells respond to GnRH-II treatment in relation to their GnRHR content. More importantly, to evaluate whether GnRH-II acts in concert with EGF to enhance the invasiveness of ovarian cancer, we cotreated all three cell lines with EGF and GnRH-II, and the results imply that these two agents have an additive effect on the invasiveness of OVCAR-3 (Fig. 5A) and CaOV-3 cells (Fig. 5B), whereas GnRH-II has no additive effect on the EGF-induced invasiveness in SKOV-3 cells (Fig. 5C). In addition, we confirmed that the expression of GnRHR was not regulated by GnRH-II (Fig. 5E) treatment but found that a small but consistent transient increase in GnRHR levels after EGF treatment only in OVCAR-3 and CaOV-3 cells (Fig. 1D), suggesting that EGF not only up-regulates GnRH-II mRNA levels but also transiently increases the GnRHR levels, which would likely further enhance EGF/GnRH-II-induced invasion in ovarian cancer cells.

Fig. 5.

Fig. 5.

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GnRH-II acts additively with EGF to promote ovarian cancer cell invasion. OVCAR-3 cells (A) CaOV-3 cells (B), and SKOV-3 cells (C) were treated with 100 ng/ml of EGF, 10 nm of GnRH-II, or in combination for 24 h and then seeded into Matrigel-coated transwells and cultured for 48 h. Noninvading cells were wiped from the upper side of the filter, and nuclei of invading cells were stained with Hoechst 33258. Left panel shows representative photos of invasion assay; right panel shows summarized quantitative results. Results are expressed as mean ± sem of at least three independent experiments. *, P < 0.05 compared with untreated control (Ctrl). #, P < 0.05 compared with EGF or GnRH-II treatment. D, The endogenous expression of GnRHR and EGFR in OVCAR-3, CaOV-3, and SKOV-3 cells were examined by Western blotting. E, OVCAR-3 cells and CaOV-3 cells were treated with 10 nm of GnRH-II for 2, 4, 8, 16, or 24 h. Cells were harvested and protein extracts were subjected to Western blotting and probed for GnRHR or β-actin as a normalization control.

EGF-induced GnRH-II production enhances the invasiveness of ovarian cancer cells

To further explore the possibility that EGF-induced synthesis of GnRH-II acts in an autocrine manner to increase the invasiveness of ovarian cancer cells, an siRNA approach was used to knock down the endogenous levels of GnRHR in OVCAR-3 cells and CaOV-3 cells. The transfected cells were then treated with 100 ng/ml EGF or 10 nm GnRH-II for 24 h before an invasion assay. This demonstrated that depletion of GnRHR in OVCAR-3 (Fig. 6A) and CaOV-3 (Fig. 6B) cells inhibited the GnRH-II-induced invasion as compared with cells transfected with a scrambled siRNA control. More importantly, the siRNA-mediated knockdown of GnRHR levels in these two cell lines also partially abolished EGF-induced invasion, further confirming that GnRH-II/GnRHR signaling is involved in the EGF-induced invasion of OVCAR-3 and CaOV-3 cells.

Fig. 6.

Fig. 6.

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GnRH-II signaling is involved in EGF-induced ovarian cancer cell invasion. OVCAR-3 cells (A) and CaOV-3 cells (B) were transfected with 100 nm type I GnRH receptor siRNA (si-GnRHR) or 100 nm siRNA controls (si-Ctrl), respectively, and followed by 100 ng/ml EGF or 10 nm GnRH-II treatment. Treated cells were then seeded into Matrigel-coated transwells and cultured for 48 h. Noninvading cells were wiped from the upper side of the filter, and nuclei of invading cells were stained with Hoechst 33258. Left panel shows representative photos of the invasion assay; right panel shows summarized quantitative results. Results are expressed as mean ± sem of at least three independent experiments. *, P < 0.05 compared with untreated control (Ctrl). #, P < 0.05 compared with EGF or GnRH-II treatment. The efficiency for GnRHR siRNA was tested by Western blotting.

Discussion

It is becoming increasingly apparent that GnRH-II acts as an autocrine/paracrine regulator in nonpituitary tissues in addition to its role in the regulation of gonadotropin synthesis and steroid hormone production (27, 28) and is an important player in cancer cell biology (6). Ovarian cancer cells also express the EGFR, and EGF is a critical mitogen involved in the differentiation of normal ovarian surface epithelial cells and the motility of ovarian cancer cells (16, 29). In the present studies, we demonstrate, for the first time, that GnRH-II expression is regulated by EGF activation of its receptor and the ERK1/2/p-CREB/C/EBPβ intracellular signaling pathway in an ovarian cancer cell model. More importantly, we have obtained evidence that EGF-stimulated GnRH-II expression constitutes a specific autocrine/paracrine loop that contributes to ovarian cancer motility.

As observed by others (30, 31, 32), EGFR activation in OVCAR-3 cells stimulates the classical MAPK/ERK1/2 pathways. Treatment of OVCAR-3 cells with EGF elicits the autophosphorylation of the EGFR at tyrosine 992 and tyrosine 1045, whereas tyrosine 992 is a target of the MAPK cascade. It is known that activation of the MAPK cascade and the subsequent phosphorylation and translocation of ERK1/2 activates transcription factors, including nuclear factor-κB, HIF-1α, and CREB, resulting in increases in the transcription of proinvasive genes such as VEGF or COX-2 (33, 34, 35). In our experiments, we have found that pretreatment of OVCAR-3 cells with the EGFR inhibitor, AG1478, abolishes EGF/EGFR downstream signaling, and that this inhibits the EGF-induced activation of a GnRH-II promoter and increases GnRH-II mRNA levels. Interestingly, EGF treatment of OVCAR-3 cells leads to increased ERK1/2 phosphorylation for up to 2 h, and others have noted that such a prolonged phosphorylation of ERK1/2 results in its increased nuclear retention (36, 37, 38). Thus, it is plausible that EGF treatment of OVCAR-3 cells prolongs the nuclear retention of activated ERK and leads to an up-regulation of GnRH-II expression in ovarian cancer cells.

It is widely accepted that CREB plays a critical role in GnRH-II expression through the transcriptional activation of the GnRH-II promoter (7). In this study, we present evidence that EGF treatment causes phosphorylation of CREB at serine133 and increases its interaction with C/EBPβ, and that these steps are required for recruitment of the transcriptional coactivator CBP (39, 40), as well as the transcriptional activation of the GnRH-II promoter. In OVCAR-3 cells, treatment with EGF enhanced CREB phosphorylation for up to 8 h, and this is correlated with the loading of p-CREB at the CRE within GnRH-II promoter, and an increase in GnRH-II promoter activity and GnRH-II mRNA levels, which are both maximal at 16 h to approximately 24 h. It is also known that the phosphorylation of ERK can lead to the activation of 90-kDa ribosomal S6 kinase and mitogen- and stress-activated kinase signaling and thereby stimulate the translocation of phosphorylated CREB (24) or phosphorylated C/EBPβ (41, 42). It was therefore of interest that blockade of EGF-induced ERK1/2 activation with a selective MAPK inhibitor (PD98059) was sufficient to block the phosphorylation of CREB, as demonstrated by Western blotting, as well as the transcriptional activation of a GnRH-II promoter-driven luciferase reporter gene (data not shown) in OVCAR-3 cells. In addition, treatment with another potent ERK1/2 inhibitor (U0126) markedly reduced the EGF-induced interaction of p-CREB with C/EBPβ in OVCAR-3 cells. Thus, it appears that ERK activation is required for the phosphorylation of CREB and its interaction with C/EBPβ, and this will contribute to its subsequent effects on the transcriptional activation of the GnRH-II gene after treatment of ovarian cancer cells with EGF.

The GnRH-II gene is regulated by several cis-acting elements within its promoter sequence (43), including an CRE (agacgtca) at nucleotide sequence −860 to −853 bp relative to the transcription start site in the GnRH-II promoter, which responds to cAMP analogs in human TE671 neuroblastoma cells (44) and human reproductive cancer cells (7). This CRE from the GnRH-II promoter has been shown to bind p-CREB/CBP/C/EBPβ under 8-bromo cAMP stimulation (7). Currently, our studies suggest that this CRE is one of the key cis-acting elements that respond to EGF stimulation. This was verified when this CRE was mutated within the GnRH-II promoter and resulted in a blockade of EGF-induced GnRH-II promoter activity. Furthermore, we used a ChIP assay to demonstrate the mechanisms of EGF-regulated CRE-mediated effects within the GnRH-II promoter. In this context, EGF-induced tethering of p-CREB at the CRE region of the GnRH-II promoter in OVCAR-3 cells, whereas loading of p-CREB onto the CRE of the GnRH-II promoter in unstimulated cells was minimal: a finding that supports a dynamic model for p-CREB association with the GnRH-II promoter after EGF stimulation. The critical importance of p-CREB in mediating EGF-induced increases in GnRH-II promoter activity in OVCAR-3 cells was further demonstrated in specific knockdown experiments, which further support the concept that p-CREB is a critical component that assembles at the GnRH-II promoter CRE after the EGF treatment of OVCAR-3 cells.

It is known that EGF and EGF-like peptides including TGF-α and amphiregulin are present in the majority of human ovarian carcinoma cells (10, 45, 46, 47). The ultimate goal of this study was to determine whether the regulation of GnRH-II by EGF is physiologically relevant to ovarian cancer cell invasiveness. Our initial experiment demonstrated that EGF induced GnRH-II mRNA levels in all three ovarian cancer cell lines (OVCAR-3, CaOV-3, and SKOV-3), and we wanted to know whether this increase of GnRH-II expression enhanced EGF-induced invasiveness in these cell lines. In support of this, we found that treatment with exogenous GnRH-II acts additively with EGF to promote the invasiveness of OVCAR-3 and CaOV-3 cells but not SKOV-3 cells. We also confirmed that neither GnRH-I nor GnRH-II have any effect on SKOV-3 cell invasiveness (6), and we attributed this to the low GnRHR levels in SKOV-3 cells as compared with OVCAR-3 and CaOV-3 cells. Moreover, because the levels of GnRHR correlate with cancer grading and are elevated in advanced stage (stages III and IV) as compared with early stage (stages I and II) ovarian carcinomas (48), our findings support the clinical data and that GnRH-II promotes the EGF-induced invasiveness of ovarian cancer cells, and further corroborate the view that GnRH-II/GnRHR plays a crucial role in tumor progression/metastasis (5, 6).

The EGFR is expressed in 33–75% of ovarian tumors. It is also frequently amplified and/or overexpressed in ovarian cancer cells, when compared with normal ovarian surface epithelial cells, and transfection with an antisense construct of EGFR into human ovarian cancer cell lines suppresses their malignant phenotype (49, 50, 51). Among the three ovarian cancer cell lines we tested, SKOV-3 cells expressed the most EGFR whereas OVCAR-3 cells expressed the least EGFR; this corresponds with the fact that SKOV-3 cells have the highest basal invasiveness whereas OVCAR-3 is the least invasive cell line. In addition, exogenous GnRH-II treatment acts additively with EGF to promote the invasive properties of OVCAR-3 and CaOV-3 cells, and GnRHR appears to be essential for this effect because siRNA-mediated down-regulation of the GnRHR completely blocked it. The down-regulation of the endogenous GnRHR also partially reduced the EGF-induced invasion in OVCAR-3 and CaOV-3 cells, and this supports our hypothesis that GnRH-II signaling is involved in the EGF-induced invasiveness of ovarian cancer cells. More importantly, EGF induced a transient increase in GnRHR levels in OVCAR-3 and CaOV-3 cells, and this could serve to further enhance the EGF/GnRH-II-induced invasion in these two cell lines. By contrast, EGF treatment did not affect the GnRHR levels in SKOV-3 cells, thus confirming our results that there is no additive effect by EGF and GnRH-II on SKOV-3 cells invasion.

In summary, our studies provide important insights into the molecular mechanism and physiological relevance of EGF-mediated GnRH-II expression in ovarian cancer. In this scenario (Fig. 7), we propose that EGF stimulation of ovarian cancer cells results in the autophosphorylation of the EGFR and induces ERK1/2 signaling, which subsequently enhances the phosphorylation of CREB and its binding with C/EBPβ. The dynamic tethering of p-CREB and C/EBPβ onto a CRE within the GnRH-II promoter then increases its transcriptional activity and results in increased GnRH-II mRNA levels in ovarian cancer cells. This ultimately enhances their production and secretion of GnRH-II, which then participates in an autocrine/paracrine loop together with EGF to promote the invasiveness of ovarian cancer cells.

Fig. 7.

Fig. 7.

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Proposed model for EGF-induced GnRH-II synthesis contributes to ovarian cancer cell invasion. EGF acting on its receptor induces the autophosphorylation of EGFR (1 ) thereby stimulating the phosphorylation of ERK1/2 (2 ). p-ERK1/2 then translocates into the nucleus (3 ) and mediates the phosphorylation of CREB, the recruitment of C/EBPβ and the binding of p-CREB onto the CRE site of the GnRH-II promoter (4 ). Up-regulated GnRH-II transcription then increases GnRH-II synthesis (5 ). Increased production of GnRH-II acts in an autocrine manner through the GnRHR (6 ), the levels of which may also be increased by EGF treatment, to stimulate the invasive potential of ovarian cancer cells (7 ).

Materials and Methods

Cells and cell culture

The human ovarian adenocarcinoma cell lines, OVCAR-3, CaOV-3, and SKOV-3, were obtained from The American Type Culture Collection (Manassas, VA). The cells were maintained in M199/MCDB105 (Invitrogen, Inc., Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS; Hyclone Laborataries Inc., Logan, UT). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2. The cells were subcultured when they reached about 90% confluence using a trypsin/EDTA solution (0.05% trypsin, 0.5 mm EDTA).

Antibodies and reagents

The polyclonal β-actin antibody and polyclonal C/EBPβ antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal phospho-ERK1/2 (Thr202/Tyr204) antibody, polyclonal total ERK1/2 antibody, polyclonal total EGFR, and monoclonal p-CREB (Ser133) antibody were obtained from Cell Signaling Technology (Danvers, MA). The monoclonal GnRHR antibody was obtained from Neomarkers (Fremont, CA), horseradish peroxidase-conjugated goat antimouse IgG and goat antirabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA). Horseradish peroxidase-conjugated donkey antigoat IgG was obtained from Santa Cruz. GnRH-II analog (d-Arg6-Azagly10-GnRH-II) was purchased from Bachem (Belmont, CA). Human EGF and EGFR inhibitor (AG1478) were obtained from Sigma Chemical Co. (St. Louis, MO). MAPK inhibitor (PD98059 and U0126) was obtained from Calbiochem (San Diego, CA).

Plasmid construction and reporter gene assays

The GnRH-II promoter-driven luciferase reporter gene construct was generated by PCR amplification of human genomic DNA using sequence-specific primers designed to amplify 2 kb upstream of the 5′-flanking region in GnRH-II promoter and followed by its subsequent cloning into the promoter-less pGL2-Basic vector (Promega Corp., Madison, WI) (8). Mutation of the CRE within the GnRH-II promoter was generated using the GnRH-II promoter-driven luciferase construct as template by the QuikChange II XL Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA), and the following oligonucleotide primers: forward, 5′-CTCTCTTCCCCTCTGAAGATACCACTGGAGTCTGGGGGTG and reverse, 5′-CACCCCCAGACTCCAG-TGGTATCTTCAGAGGGGAAGAGAG. The product was sequenced to verify that only the desired mutation had occurred during the mutagenesis reaction.

Transient transfections were carried out using Lipofectamin 2000 Reagent (Invitrogen, Inc., Burlington, Ontario, Canada) following the manufacturer’s protocol. To correct for transfection efficiencies, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into the cells with the GnRH-II promoter-driven luciferase reporter gene construct. Briefly, 5 × 105 cells were seeded into six-well tissue culture plates the day before transfection. The GnRH-II promoter-driven luciferase reporter gene construct (1 μg) and 0.5 μg RSV-lacZ plasmid were cotransfected into cells grown in standard culture medium containing FBS. In some experiments, 150 nm small interfering CREB or a control siRNA oligonucleotide (QIAGEN, Inc., Mississauga, Ontario, Canada) were cotransfected with the reporter plasmids. After 6 h, 2 ml of serum free medium was added, and the cells were further incubated overnight (18 h). The culture medium was then removed and the cells were treated with EGF or 8-bromo cAMP, respectively, in serum free medium for the times indicated. Cellular lysates were collected with 150 μl reporter lysis buffer (Promega) and assayed for luciferase activity. The β-Galactosidase Enzyme Assay System (Promega) was used to measure expression from the RSV-lacZ plasmid, and promoter activities were expressed as luciferase activity/β-galactosidase activity.

Real-time PCR

After treatment with 8-bromo cAMP, medium was removed from the culture dish, and RNA was extracted using Trizol (Invitrogen). The RNA concentration was measured based on the absorbance at 260 nm, and its integrity was confirmed by agarose-formaldehyde gel electrophoresis. Total RNA (2.5 μg) was reverse-transcribed into cDNA using a first-strand cDNA synthesis kit (GE Healthcare Bioscience, Piscataway NJ) following the manufacturer’s procedure. The primers used for SYBR Green real-time RT-PCR were designed using Primer Express Software version 2.0 (Applied Biosystems, Foster City, CA). The primers for GnRH-II mRNA are: sense, 5′-CTGCTGACTGCCCACCTT; and antisense, 5′-GCTTTCCTCCAGGGTACCAG. The primers for glyceraldehyde-3-phosphate dehydrogenase are: sense, 5′-GAGTCAACGGATTTGGTCGT; and antisense, 5′-GACAAGCTTCCCGTTCTCAG. Real-time PCR was performed using the ABI prism 7000 Sequence 10 Detection System (Applied Biosystems) equipped with a 96-well optical reaction plate. The reactions were set up with 16.5 μl SYBRR Green PCR Master Mix (Applied Biosystems). All real-time experiments were run in triplicate, and a mean value was used for the determination of mRNA levels. Negative controls, containing water instead of sample cDNA, were used in each experiment. Relative quantification of the mRNA levels for GnRH-II in ovarian cancer cells was performed using the comparative CT method with glyceraldehyde-3-phosphate dehydrogenase as an endogenous control and with the formula 2−ΔΔCt.

Nuclear protein extraction, Western blotting, and immunoprecipitation

Briefly, cells were washed with ice-cold PBS and harvested with 1 ml solution A (10 mm HEPES, pH 7.9; 10 mm KCl; 10 mm EDTA; 0.5 mm dithiothreitol; 1 μg/ml aprotinin; and 1 μg/ml protein inhibitor cocktail). Cell lysates were transferred to 1.5-ml centrifuge tubes and placed in an orbital rocker for 10 min at 4 C. Nuclear pellets were obtained by centrifugation at 14,000 × g at 4 C for 10 min, and supernatants were collected for cytoplasmic protein. Nuclear pellets were resuspended in solution B (100 mm HEPES, pH 7.9; 2 m NaCl; 5 mm EDTA; 50% Glycerol) and placed in an orbital rocker for 2 h at 4 C. After centrifugation at 14,000 × g at 4 C for 5 min, supernatants containing the nuclear protein extracts were removed. The nuclear extracts were then subjected to electrophoresis on an 8% SDS-PAGE gel and Western blotted for detection with appropriate antibodies.

Immunoprecipitation was conducted as follows: nuclear extracts were incubated with p-CREB antibody (10 μg/ml) followed by the antibody capture affinity ligand provided by the immunoprecipitation kit (Upstate Biotechnology, Inc., Lake Placid, NY) at 4 C overnight. The immunoprecipitated proteins were then subjected to electrophoresis on an 8% SDS-PAGE gel and detected with appropriate antibodies after Western blotting.

ChIP

All reagents, buffers, and supplies were included in a ChIP-IT kit (Active Motif, Inc., Carlsbad, CA). Briefly, the cells were cross-linked with 1% formaldehyde for 10 min at room temperature. After washing and treatment with glycine Stop-Fix solution, the cells were resuspended in lysis buffer and incubated for 30 min on ice. The cells were homogenized, and nuclei were resuspended in shearing buffer and subjected to preoptimized ultrasonic disruption conditions to yield 100- to 500-bp DNA fragments. The chromatin was precleared with Protein G beads and incubated (overnight at 4 C) with 1 μg of the following antibodies: negative control mouse IgG (Active Motif), p-CREB antibody (Cell Signaling Technology). Protein G beads were then added to the antibody/chromatin incubation mixtures and incubated for 1.5 h at 4 C. After extensive washing, the immunoprecipitated DNA/protein complex was removed from the beads by elution buffer. To reverse cross-links and remove RNA, 5 m NaCl and ribonuclease were added to the samples and incubated at 65 C for 4 h. The samples were then treated with proteinase K for 2 h at 42 C, and the DNA was purified using gel exclusion columns. The purified DNA was subjected to PCR amplification (one cycle of 94 C for 3 min; 40 cycles of 94 C for 20 sec; 64 C for 30 sec and 72 C for 30 sec) for the CRE site (−860/−853 bp) within the GnRH-II promoter using specific forward, 5′-CCAGCCTAAAGCAAGAGTCC and reverse, 5′-GTCTATAAATCCTGGGGCCA primers. As an input control, 10% of each chromatin preparation was used. The PCR products (213 bp) were resolved by electrophoresis in a 2.5% agarose gel and visualized by ethidium bromide staining (7). The ChIP assay was performed at least three times, and consistent data were obtained between experiments.

Invasion assay

The invasion assay was performed in Boyden chambers with minor modifications (52). Filters (8-μm pore size, 24 wells; BD Biosciences, Palo Alto, CA) were coated with 40 μl of 1 mg/ml growth factor-reduced Matrigel (BD Biosciences). Cells in M199/MCDB105 medium supplemented with 0.1% FBS were incubated for 48 h against a gradient of 10% FBS for OVCAR-3 cells and 5% FBS for CaOV-3 and SKOV-3 cells. Cells that penetrated the membrane were fixed with ice-cold methanol and stained with Hoechst 33258 (Sigma), and the number of nuclei was counted using Northern Eclipse 6.0 software from Empix Imaging (Mississauga, Ontario, Canada). Each treatment was done in duplicate, and five microscopic fields were counted per Boyden chamber.

Data analysis

Reporter gene assays and real-time PCR data are shown as the mean ± sem of three independent experiments. Data were analyzed by one-way ANOVA, followed by Tukey test using the computer software PRISM (GraphPad Software, Inc., San Diego, CA). Values were considered significantly different from each other at P < 0.05.

Acknowledgments

We thank Dr. Geoffrey L. Hammond for critical reading of the manuscript.

Footnotes

This work was supported by an operating grant from the Canadian Institutes of Health Research (to P.C.K.L.). P.C.K.L. is recipient of a Child & Family Research Distinguished Scholar Award. S.L.P. was the recipient of graduate studentship awards from Cordula and Gunter Paetzold Fellowship.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 16, 2009

Abbreviations: CBP, CREB-binding protein; C/EBPβ, CCAAT/enhancer binding protein β; ChIP, chromatin immunoprecipitation; CRE, cAMP-responsive element; CREB, cAMP-responsive binding protein; EGF, epidermal growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; GnRHR, GnRH receptor; p-CREB, phosphorylated CREB; p-ERK1/2, phosphorylated ERK1/2; RSV, Rous sarcoma virus; siRNA, small interfering RNA.

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